The human CRC cell lines SW480, HT29, HCT-8, LoVo and HCT116 were purchased from the American Type Culture Collection (Manassas, VA, USA), whereas the human normal colon mucosal epithelial cell line NCM460 was obtained from INCELL Corporation LLC (San Antonio, TX, USA). The cell culture media of McCoy's 5A, RPMI-1640, F12-K, Leibovitz's L-15 and DMEM were purchased from Gibco (Thermo Fisher Scientific, Inc.). HCT116 and HT29 cells were cultured in McCoy's 5A medium. HCT-8, LoVo, SW480 and NCM460 cells were maintained in RPMI-1640, F12-K, Leibovitz's L-15 and DMEM, respectively. All cell culture media aforementioned were supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and streptomycin. The cells were incubated under 5% CO₂ at 37°C.

Human hsa-miR-139-5p mimics (micrON™ hsa-miR-139-5p mimics; cat. no. miR10000250-1-5) and miRNA negative control (NC) mimics (micrON™ mimics Negative Control #24; cat. no. miR01201-1-5) were synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). The cells were respectively seeded in 24-well plates at a density of 5×10⁴ cells/well and then transfected with 60 nM miR-139-5p mimics or NC mimics at 60% confluence using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to manufacturer's protocol. The cells transfected with the miR-139-5p or NC mimics were harvested at 48 h post-transfection.

Total RNA was extracted from the cell pellets using the TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to manufacturer's protocol and subsequently quantified using NanoDrop™ 2000 machine (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. For miRNA detection, the TaqMan MicroRNA assay kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used according to the protocol supplied by the manufacturer. The expression level of miR-139-5p was normalized to that of the internal control, U6. For mRNA detection, the synthesis of cDNA was performed using the PrimerScript™ RT Reagent kit (Takara Bio Inc.) and qPCR was conducted by the FastStart Universal SYBR Green Master kit (Roche Diagnostics), according to the manufacturer's protocol. The mRNA expression levels of IL-6, IL-1β and TNF-α were normalized to that of GAPDH, which was used as an internal control. The primer sequences were listed in Table I. The thermal cycling conditions for PCR were: Initial denaturation at 95°C for 15 min, followed by 35 cycles of denaturing at 95°C for 15 sec, annealing at 55°C for 30 sec and elongation at 72°C for 30 sec. The results of the detection were calculated using the 2^−ΔΔCq analysis method (18).

The effect of miR-139-5p on cell proliferation was evaluated using the Cell Counting Kit-8 (CCK-8; Sangon Biotech Co., Ltd.). Briefly, cells transfected with the miR-139-5p or NC mimics were plated into the 96-well plates (2×10³ cells/well), and 10 µl CCK-8 solution was added to each well following culturing for 24, 48 and 72 h. The plate was then incubated at 37°C for 4 h, and cell proliferation was evaluated by measuring the absorbance at 450 nm using Multiskan MS (Thermo Fisher Scientific, Inc.). A total of three wells were used in each group, and the experiments were repeated three times independently. The results are expressed as the mean values ± standard deviation (SD).

The cells were seeded in a 24-well plate (5×10⁴ cells/well) and transfected with miR-139-5p or NC mimics. After 48 h of incubation, the cells were collected and seeded (1,000-1,500 cells/well) into a fresh 6-well plate for 10 days. The surviving colonies were counted following fixation with methanol/acetone (1:1) and were finally stained with 0.5% methylene blue (MedChemExpress). The experiments were repeated three times.

Cell invasion was examined by a Matrigel invasion assay (BD Biosciences). In brief, the cells were transfected with the miR-139-5p or NC mimics for 48 h, and then harvested and suspended in serum-free medium. A total of 1×10⁵ cells were diluted in 500 µl serum-free medium and added to the upper chamber of the Transwell apparatus (Corning Inc.) that was precoated with 1 mg/ml Matrigel (BD Biosciences) for 2 h at 37°C Subsequently, 0.6 ml serum-free media with 10 ng/ml hepatocyte growth factor was added to the lower chamber. After 48 h of incubation, the cells that invaded through the Matrigel membrane were fixed with 4% paraformaldehyde at room temperature for 30 min, stained with 0.5% crystal violet at room temperature for 30 min and counted in five high-power fields (magnification, ×200). A total of three independent experiments were conducted.

The total protein was extracted from the cells using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology) with proteinase inhibitor cocktail (Roche Diagnostics). Subsequently, the protein concentration was quantified using the BCA Protein Assay Reagent kit (Thermo Fisher Scientific, Inc,) and ~30 µg total protein was then separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (EMD Millipore; Merck KGaA). Non-specific binding was blocked by incubating the membranes in 5% skim milk for 1 h at room temperature. Next, the membranes were incubated overnight at 4°C with rabbit anti-matrix metalloproteinase 9 (MMP9, cat. no. 13667), rabbit anti-MMP7 (cat. no. 3801) and rabbit anti-GAPDH (cat. no. 5174) monoclonal antibodies (dilution, 1:1,000; all purchased from Cell Signaling Technology, Inc., Danvers, MA, USA). Following washing, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies (cat. no. 5127; 1:10,000, Cell Signaling Technology, Inc.) at room temperature for 30 min and visualized using the automatic chemiluminescence image analysis system (Tanon 6100; Tanon Science & Technology Co., Ltd.).

The cells were transfected with miR-139-5p or NC mimics, and then seeded in 24-well plates (5×10⁴ cells/well). After 48 h of incubation, the cell culture supernatant was collected from the 24-well plates, and the content of the inflammatory cytokines, including IL-1β, IL-6 and TNF-α, was measured by ELISA kits (Sangon Biotech Co., Ltd.) according to the protocols supplied the manufacturer. A total of three independent experiments were performed in triplicate.

An electrophoretic mobility shift assay (EMSA) was performed to assess NF-κB activation, as previously described (19). Briefly, HCT116 cells were transfected with miR-139-5p or NC mimics for 48 h prior to harvest. The nuclear extract was prepared using a Nuclear Extract kit (EpiGentek Group, Inc.). An EMSA kit (Thermo Fisher Scientific, Inc.) was used for detection of DNA binding, and DNA fragments were synthesized as described previously (20). Subsequently, the complexes of NF-κB with the DNA were separated on 4% non-denaturing polyacrylamide gel electrophoresis and transferred to PVDF membranes. The membranes were exposed to UV light (120 mJ/cm²) at room temperature for 10 min and then probed with a streptavidin-horseradish peroxidase conjugate (cat. no. S911, Thermo Fisher Scientific, Inc.). The membranes were then wrapped in plastic film were placed in the middle of two X-ray films, placed together in a cassette and developed at −80°C for 72 h. The bands were analyzed using an ImageJ Software (version 1.8.0; National Institutes of Health).

A xenograft mouse model was used to evaluate the effects of miR-139-5p in regulating tumor growth. The experimental protocol involving animals was approved by the Animal Ethics Committee of the Kunming Medical University (Kunming, China). Briefly, specific pathogen-free female BALB/c nude mice (n=9), aged 4–6 weeks old and weighing 20–30 g, were obtained from the Guangdong Medical Laboratory Animal Center (Foshan, China). Mice were housed in pathogen-free room at 24±2°C, with 60–80% humidity and had free access to food and water with a 12-h light/dark cycle. The animals were divided into three groups (3 animals/group), as follows: Blank, NC mimics and miR-139-5p mimics groups. Transfected HCT116 cells were injected subcutaneously into the left flank of the mice (5×10⁶ cells per mouse), respectively. The tumor size was measured every 4 days using a vernier caliper, and the tumor volume was calculated as follows: Volume=(length × width × width)/2. The animals were sacrificed by cervical dislocation on day 28, and the tumors were excised and snap-frozen.

The data are presented as the mean ± SD. All experiments were repeated at least three times with duplicate or triplicate samples in each assay. The statistical differences were evaluated by conducting Student's t-test and one-way analysis of variance using the SPSS software (version 22.0; IBM Corp.). A P-value of <0.05 was considered to be an indicator of a statistically significant difference.

Previous studies have reported that the expression levels of miR-139-5p are downregulated in CRC tissues and are associated with poor disease prognosis (15,21). Although miR-139-5p shares the same precursor with miR-139-3p, miR-139-3p expression has been reported to be undetectable in CRC cells, the overexpression of which exhibited no significant effects on the malignant behavior of CRC cells (15). Therefore, in the present study, the function of miR-139-5p was examined. The data revealed significant downregulation of miR-139-5p in five human CRC cell lines, including SW480, HT29, HCT-8, LoVo and HCT116 cells, compared with its expression in the normal human colon mucosal epithelial cell line NCM460 (Fig. 1A). This observation suggests that miR-139-5p may serve an important role in CRC tumorigenesis. In accordance to these findings, a previous study has reported that miR-139-5p sensitized LoVo and HCT-116 cells to 5-fluorouracil via NOTCH-1, with further increase in apoptosis and reduced viability observed in these cells (22), indicating that miR-139-5p may serve an important role in LoVo and HCT-116 cells. Thus, these two cell lines were selected for the further investigation in the present study.

To investigate the effects of miR-139-5p on the proliferation and invasion of CRC cells, miR-139-5p mimics were transfected into CRC cells (HCT116 and LoVo), and RT-qPCR was used to determine the expression levels of miR-139-5p. The results indicated an increased expression of miR-139-5p in HCT116 and LoVo cells following transfection with miR-139-5p mimics, whereas NC mimics had no marked effect compared with the blank group (Fig. 1B). It was observed that ectopic expression of miR-139-5p significantly inhibited the proliferation of HCT116 and LoVo cells when compared with the blank group (Fig. 2A and B). The inhibitory effects of miR-139-5p on colon cancer cell proliferation were further confirmed by the colony formation assay. The results indicated that overexpression of miR-139-5p markedly suppressed the colony formation ability in miR-139-5p mimic-transfected HCT116 and LoVo cells compared with that noted in the blank or NC mimics groups (Fig. 2C). Subsequently, the inhibitory effects of miR-139-5p on tumor growth were further confirmed in vivo by conducting a xenograft tumor growth assay. The subcutaneous tumor growth curve is shown in Fig. 2D and indicates that the tumor growth rate of HCT116 cells transfected with miR-139-5p mimics was significantly lower compared with that of the blank and NC mimics cells on day 28. These results provided further evidence that miR-139-5p serves a tumor suppressive role in colon cancer.

Following the initial results of miR-139-5p on colon cancer cell growth, the current study further investigated whether this miRNA affects the invasiveness of colon cancer cells in vitro. The number of invading cells in the miR-139-5p mimic-transfected HCT116 and LoVo cells were significantly decreased compared with those in the blank or NC mimic-transfected groups (Fig. 3A). The reduction in the number of invading cells caused by the overexpression of miR-139-5p further confirmed the effect of this miRNA in suppressing cell invasion.

The MMP enzymes comprise a family of extracellular proteinases enzymes that regulate basic cellular processes, such as cell invasion. Two main members, namely MMP9 and MMP7, have been demonstrated to play significant roles in cell invasion (23,24). Therefore, the levels of MMP9 and MMP7 were measured by western blot assays in the current study. The data demonstrated that overexpression of miR-139-5p evidently inhibited the protein expression levels of MMP7 and MMP9 (Fig. 3B). Taken collectively, these results strongly indicated that miR-139-5p overexpression inhibited cell invasion.

The inflammatory response contributes to malignant tumor progression in the tumor microenvironment (25,26). Changes in the inflammatory microenvironment are frequently accompanied by molecular alternations in tumor tissues, and miRNAs are usually considered as potential mediators of these processes (27). In the present study, overexpression of miR-139-5p significantly decreased the mRNA expression levels of the inflammatory cytokines IL-1β, IL-6 and TNF-α in HCT116 and LoVo cells transfected with miR-139-5p mimics (Fig. 4A). Furthermore, transfection of the cells with miR-139-5p mimics significantly decreased the secretion of IL-1β, IL-6 and TNF-α in the culture supernatant of HCT116 and LoVo cells (Fig. 4B). It is well known that IL-1β, IL-6 and TNF-α are target genes of NF-κB, and its activation has been demonstrated to boost the production of these inflammatory cytokines (28). The results of the present study indicated that activation of NF-κB was apparent in HCT116 cells, whereas it was considerably suppressed by overexpression of miR-139-5p in these cells (Fig. 4C and D). Taken collectively, the data suggested that miR-139-5p was able to decrease the production of IL-1β, IL-6 and TNF-α by suppressing NF-κB activation.